MAN0001771 Revised: 24May2010 | G002

Table 1. Contents and storage information.

Product Amount Concentration Storage Stability
Please refer to product label
20C
Desiccated
Protected from light
or
20C
Desiccated
(see the label)
When stored as directed,
product is stable for 6 months.
Acetoxymethyl (AM) and Acetate Esters
Introduction
The acetoxymethyl (AM) ester derivatives of fluorescent indi ca tors and chelators make up
one of the most useful groups of compounds for the study of live cells. Modifica tion of carbox-
y lic acids with AM ester groups results in an uncharged molecule that can permeate cell
membranes. Once inside the cell, the lipophilic blocking groups are cleaved by nonspecific
ester ases, resulting in a charged form that leaks out of cells far more slowly than its parent
compound. Frequently, hydro lysis of the esterified groups is essential for binding of the target
ion. In some cases (e.g., calcein AM), the AM ester is color less and non fluo rescent until
hydrolyze d. This property is useful in diag nosing spontaneous hydrolysi s during storage.
Acet ate groups, used on many fluorescent indicators, are analogous to AM ester groups and
should be treated similarly.
Guidelines for Use
Preparing the Stock Solutions AM or acetate esters should be reconstituted only as require d using high-quality, an hydrous
dimethylsulfoxide (DMSO). Reagent-grade DMSO should be stored well sealed under argon
or nitro gen, and desic cated; desic cant beads (e.g., molecular sieves) can be used for short-
term stor age. Dissolution of the pure AM or acetate ester s in DMSO may be slow (particularly
in the 1 mg sizes). Once prepared, DMSO stock solutions of AM or acetate esters should
preferably be used within a short time period for one series of experi ments. DMSO stock
solutions must be kept anhydrou s, since the solvent will readily take up moisture, leading to
decomposition of the dye. These stock solutions should be stored well sealed, frozen, and
desiccat ed. Many of our AM and acetate esters are available in small ali quots. Use of the
pre aliquoted product is strongly recommended.
It is advisable to keep the AM ester or acetate ester in as concen trated a stock as possible so that
minimal amounts (ideally 0.1%) of DMSO are present in the loading solution.
Acetoxymethyl (AM) and Acetate Esters | 2
Using Pluronic F-127
Dispersing Agent Since some AM esters (particularly SBFI, AM and PBFI, AM) are relatively insoluble in aque-
ous solutions, the low-toxicity dispers ing agent Pluronic F-127 is often used to facilitate cell
loading. This nonionic detergent can be made up to a final concentration of 20% (w/v) in
DMSO, and this solution can be used to prepare the dye stock. Gentle warming ( ~40C) may
assist in gettin g the detergent into DMSO. Pluronic F-127 may de crease the stability of AM
esters, so it should only be added to working stocks.
For the convenience of our customers, Molecular Probes offers Pluronic F-127 in three
forms: 1 mL of a 20% (w/v) solution in DMSO (P3000), 30 mL of a 0.2 mfiltered 10% (w/v)
solution in water (P6866) and 2 g solid (P6867).
Whether Pluronic is used or not, it is advisable to keep the AM ester or acetate ester in as
concen trated a stock as possible so that minimal amounts (ideally 0.1%) of DMSO are present
in the loading solution.
Loading of Cells with AM or
Acetate Esters This is intended as an introduc tion only. Specific protocols for any particular dye and cell
type should be obtained from the literature. As a rule, AM and acetate esters are used at a
final working concentration of between 1 and 10 M. Higher concentrations of weakly fluo-
rescent indicators such as Fura Red and quin-2 may be required. The AM or acetate ester
concen tration should be kept as low as possible to reduce potential artifacts from overloading,
including incomplete hydrolysis, compart men tali za tion, and toxic effects of hy drolysis by-
products such as form aldehyde or acetic acid. Generally, loading times of betwee n 15 minutes
and 1 hour are sufficient, al though probes such as SBFI, AM and PBFI, AM may require
14 hours. Loading may be done at a tem pera ture that is optimal for the cells, although some
investigators have report ed greater degrees of compart men tali za tion at physio logical tem-
peratures than at room temperature. In addition to assist ing in dye uptak e, Pluronic F-127
may help in reducing compartmentalization. To keep extracellular hydroly sis of the AM and
acetate esters to a mini mum, it is recommended that a loading buffer free of prima ry and
secondary amines such as PBS be used. Cells should be washed in dye-free buffer after loading.
Assessing Dye Responsiveness The following protocol is optional and can be used to test the responsiveness of the dye (for
example following extended storage) or as a means to calibrate the ion response of the dye.
Following the calibration or testing of the dye using this protocol, it should be disposed of
and not loaded into cells.
We generally recommend that the separately available salt or free acid form of an indicator be
used for calibrating the ion response. However, the following protocol for AM or acetate ester
hydro lysis provides a less-preferred alternative and may also be useful to assess spontaneous
hydrolysis during storage. This procedure is not always successful for AM esters, probably
because of the formation of formaldehyde in the reaction.
1.1 Dissolve a small amount of the AM or acetate ester (e.g., 50 g calcein AM) in 50 L dioxane,
DMSO, or other water-miscible solvent.
1.2 Add an equal volume of methanol.
1.3 Add 25 L of 2 M KOH/water. If the dye is not in solution at this point, add more methanol.
1.4 Wait one hour.
1.5 Adjust pH to ~7 with HCl.
Acetoxymethyl (AM) and Acetate Esters | 3
1.6 Test for fluorescent response. For exam ple, to test a calcium indi cator, dilute 5 L of
the dye solution into 100 L of water, add this sepa rately to high-calcium buffer and to low-
calcium buffer.
1.7 If the dye does not respond properly, add more KOH/metha nol to the dye solution and
repeat steps 1.6 and 1.7.
Related Products Current prices may be obtained from our website or from our Customer Service Department.
Cat # Product Name Unit Size
P6867 Pluronic F-127 *low UV absorbance* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
P3000 Pluronic F-127 *20% solution in DMSO*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mL
P6866 Pluronic F-127 *10% solution in water* *0.2 m filtered*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 mL
P10020 PowerLoad 100X concentrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mL
P36400 Probenecid, water soluble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 77 mg
Contact Information
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Fax: (541) 335-0504
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Technical Services: eurotech@invitrogen.com
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